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problem of membrane protein FPLC

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#1 handsome



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Posted 28 February 2010 - 08:42 PM

I purified my membrane protein by FPLC gel filtration column. Before the injection , I checked the OD280, which is around 1AU. then i took 0.5ml protein sample to run the FPLC. However, no sharp peak came out. I just got a broad and small peak with 35 mAu. which is much lower than that expected. (I purified the membrane protein with DM and the FPLC buffer : 150mM NaCl, 20mM HEPEs, 0.4g/100ml DM)

Is it that my protein has aggregated on the FPLC column?

I also try to increase salt concentration (300mM) of the FPLC buffer , then I got a sharp peak with OD 160 mAu and the retention time is some early than Void Volume. Is it normal ? anybody can give me some suggestion for the FPLC pufication condition?

#2 mdfenko


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Posted 02 March 2010 - 12:57 PM

which fplc column did you use?

recommended minimum ionic strength for fplc buffer (gel filtration) is 200mM. this prevents non-specific binding to the stationary phase.

if the peak came before the void then it was something released from the column prior to the injection. when you re-equilibrated with the higher salt buffer you probably did not completely wash the column before you injected (anything bound that was released will fractionate).
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